UHPLC-ESI-Q-TOF-MS/MS analysis, antioxidant activity

RSC Advances

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UHPLC-ESI-Q-TO

aSchool of Pharmacy, Shenyang Pharmac

Shenyang, Liaoning 110016, P. R. China. E-bSchool of Pharmaceutical Engineering,

Shenyang, Liaoning, P. R. China

† Electronic supplementary informa10.1039/c8ra02431f

Cite this: RSC Adv., 2018, 8, 27661

Received 20th March 2018Accepted 17th July 2018

DOI: 10.1039/c8ra02431f

rsc.li/rsc-advances

This journal is © The Royal Society of C

F-MS/MS analysis, antioxidantactivity combined fingerprints for qualityconsistency evaluation of compound liquoricetablets†

Yujing Zhang,a Chao Wang,b Fangliang Yang,a Zhe Yang,a Fangren Wanga

and Guoxiang Sun *a

Traditional Chinese medicines (TCM)/herbal medicines (HM) are too complicated to comprehensively

investigate their quality consistency effectively with a single detection technique. Hence, finding an

effective, rapid, and comprehensive quality control (QC) method is of great importance for guaranteeing

the safety and efficacy of TCM/HM in clinical applications. In our current research, a novel strategy of

multi-wavelength fusion HPLC fingerprints and ultraviolet (UV) spectroscopic fingerprinting was

proposed and successfully applied to monitor the quality consistency of compound liquorice tablets

(CLT). The quality grades of 35 CLT samples from two manufacturers were successfully discriminated

and evaluated by the averaged linear quantified fingerprint method (ALQFM) from a qualitative and

quantitative perspective. The results showed that the UV spectroscopic fingerprints agreed well with the

multi-wavelength fusion HPLC fingerprints. In addition, ultra-high-performance liquid chromatography

coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-

MS) was applied to investigate the chemical constituents in CLT samples, providing an important

chemical structural foundation for further QC and bioactivity studies. Additionally, a simple flow injection

analysis (FIA) was developed to investigate the antioxidant capacity in CLT, which was based on the

scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals by antioxidants. Furthermore, the fingerprint–

efficacy relationship between high-performance liquid chromatography (HPLC) fingerprints and the

antioxidant activities of CLT samples was established utilizing orthogonal projection to latent structures

(OPLS). In conclusion, this study indicated that integrating UHPLC-ESI-Q-TOF-MS/MS, UV spectroscopic

fingerprints, and multi-wavelength fusion HPLC fingerprints coupled with the antioxidant activities

reported could give important clues for further pharmacological and clinical studies of CLT. Meanwhile,

it provides a practical strategy for the rapid screening and identifying of TCM/HM quality consistency.

1. Introduction

TCM/HM, as alternative medicines, are becoming increasinglypopular worldwide. However, the therapeutic effects of mostTCMs/HMs are combined with others in a single prescriptionbased on the synergistic efficacies of multi-constituents towardmulti-targets.1,2 The compatibility mechanisms of herb–druginteractions have attracted much attention, which could beattributed to the content variation of the chemical compoundsin vitro.3 Clearly, it is not sufficient to just monitor the TCM/HMquality by quantifying a limited number of marker substances.

eutical University, 103 Wenhua Road,

mail: [email protected]

Shenyang Pharmaceutical University,

tion (ESI) available. See DOI:

hemistry 2018

Fingerprinting techniques, as rational and powerful methodsfor systemically characterizing complicated TCMs/HMs, havebeen widely accepted and adopted for TCM/HM QC in recentyears.4 Nowadays, commonly used ngerprints involve singlewavelength acquisition; however, this cannot reect compre-hensive chemical information of TCMs/HMs. Therefore,a multi-wavelength fusion HPLC ngerprints method5,6 hasincreasingly been adopted to realize the overall evaluation ofthe TCM/HM quality based on combining computer technologyand macro ngerprint characteristics.

In fact, various approaches for chromatographic nger-printing based on various detection techniques, such asHPLC,7–9 gas chromatography (GC),10,11 and capillary electro-phoresis (CE),12 are frequently used for TCM/HM QC; however,it should be noted that these tend to be time-consuming andlaborious; moreover, in most cases they cannot offer informa-tion about all the chemical components in the TCM/HM, which

RSC Adv., 2018, 8, 27661–27673 | 27661

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https://doi.org/10.1039/c8ra02431f

https://pubs.rsc.org/en/journals/journal/RA

https://pubs.rsc.org/en/journals/journal/RA?issueid=RA008049

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restricts the overall QC of the multi-constituents. In this situa-tion, ow-based methods, such as ow injection analysis (FIA),multisyringe FIA, and sequential injection analysis, have beenproposed and even successfully applied in automated analysisfor rapid screening purposes.13–15 On the basis of FIA, UVspectroscopic ngerprinting is preferred to monitor the TCM/HM quality, owing to its apparent analytical advantages, suchas low experimental expenses, simple sample preparation, andshort measurement time. It is common knowledge that UVspectra can display the features of unsaturated bonds infor-mation, such as for n–s*, n–p*, and p–p*, qualitatively andquantitatively;16 therefore, UV spectroscopic ngerprinting incombination with chemometric methods can be used formonitoring the TCM/HM quality in a straightforward, rapid,and effective manner.17,18 In the past few years, UHPLC–ESI-Q-TOF-MS/MS, as a high resolution, excellent sensitivity, accu-rate mass measurement and high throughput technology, hasbeen applied to integrate multi-constituent determination andngerprint analysis for TCM/HM QC,19 and could be of greatimportance for facilitating further investigation of thecompatibility mechanisms.

On the other hand, conventional chromatographic nger-print analysis is typically used for authenticity and identica-tion, but this only indicates the qualitative similarity andignores the quantitative assessment.20,21 Although, the quanti-tative evaluation of chromatographic ngerprinting in the QC ofCHM has been veried;22–24 nevertheless, multi-componentquantication is not credible in situations where certainmedicinal ingredients are ignored. In this study, both theaveraged linear qualitative and averaged quantitative similari-ties of the ngerprints of the CLT samples were assessed by theaveraged linear quantied ngerprint method (ALQFM), whichwas developed and successfully applied to address the issue ofperforming a qualitative and quantitative comparison of thereference standards and PPCE samples.25

CLT is an important TCM prepared from ve medicinalherbs, including licorice root (extract) concrete (LRC), powderedpoppy capsule extractive (PPCE), camphor, oleum anisi stellati,and sodium benzoic (SMB), which have been widely used foranti-inammatory, antitussive, expectorant, and antiasthmaticactions.26 The compositions of CLT mainly include avonoids,saponins, alkaloids, and other compounds, as reported inprevious reports,27–44 and these chemical components havea common pharmacological activity, that is, their antioxidantactivity.45–47 Moreover, the published studies have shown thatthe antioxidant components, especially avonoids, saponins,and alkaloids of herbal products, can decrease the risk ofnumerous diseases, such as inammation, senescence cardio-vascular disease, neurodegenerative disorders, diabetes, andcancer, closely related to the damaging effects of free radi-cals.48–51 Accordingly, to discover natural antioxidants and theirantioxidant properties from food and TCMs/HMs,52 the devel-opment of rapid, qualitative, and quantitative analytical tech-niques are important tasks. Stable free radical species, such as1,1-diphenyl-2-picrylhydrazyl (DPPH), are oen used for evalu-ating the free radical scavenging capacity of various antioxi-dants based on the ability of an antioxidant to quench free

27662 | RSC Adv., 2018, 8, 27661–27673

radicals by hydrogen donation.53 This encourages us to explorethe antioxidant activities to reect the bioactivity of CLT and tocorrelate them with their ngerprints.

In the present work, multi-wavelength fusion HPLC nger-printing and UV spectroscopic ngerprinting method weredeveloped for assessing the quality consistency of 35 CLTsamples, where the two methods perfectly complement eachother. In ngerprint assessments, ALQFM was established forscientic CLT quality analysis from a qualitative and quantita-tive perspective. In addition, the antioxidant capacity of CLTwas determined based on FIA; besides, the ngerprint efficacybetween a fusion ngerprint and antioxidant capacity was per-formed utilizing an OPLS model. An efficient UHPLC-ESI-Q-TOF-MS/MS was exploited to elucidate the chemical compo-nents in CLT. It was demonstrated that integrating UV spec-troscopic ngerprinting and multi-wavelength fusion HPLCngerprinting coupled with UHPLC-ESI-Q-TOF-MS/MS andassessing the antioxidant activities offers a powerful and effec-tive method for QC and can facilitate further investigation of thecompatibility mechanisms of CLTs.

2. Theory of ALQFM54

For the modeling, we assumed that the sample ngerprintvector (SFPV) and the reference ngerprint vector (RFPV) were~x ¼ ðx1; x2;/xnÞ and~y ¼ ðy1; y2;/ynÞ, respectively, where xi andyi are the ith peak area in the sample and reference ngerprints,respectively. The average linear qualitative similarity (SL),a qualitative parameter calculated by eqn (1), can accuratelydescribe the resemblance in terms of the number and distri-bution of ngerprints between the RFPV and SFPV. On the otherhand, the slope of the linear equation (b), as calculated by eqn(2), can quantitatively compare ~x and ~y aer being weight-corrected by mS and mR, where mS is the weight of eachsample and the parameter mR is the average weight of the 35CLT samples. Furthermore, the average linear quantitativesimilarity (PL), a quantitative parameter calculated by eqn (3),was revised by b to examine the total content of all the nger-print components similarity for all ingredients between SFPVand RFPV. Finally, the apparent content similarity (R%, eqn (4))and b were calculated for error evaluation of the ngerprints. Angerprint variation coefficient (a), as dened in eqn (5), wasdetermined as a statistical error that can reect the ngerprintdissimilarity between SFPV and RFPV.

Accordingly, the quality evaluationmethod in terms of SL, PL,and awas named as ALQFM, by which the TCM quality could beclassied into 8 grades (Table 1). In the evaluation system, thelower the grade values, the better the quality, where grades 1–5are recognized as qualied.

SL ¼ 1

2

0BBBB@

Pni¼1

ðxi � xiÞðyi � yiÞffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPni¼1

ðxi � xiÞ2s ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPn

i¼1

ðyi � yiÞ2s þ

Pni¼1

xi

yiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffinPni¼1

�xi

yi

�2s1CCCCA (1)

This journal is © The Royal Society of Chemistry 2018




Table 1 The quality grades assigned by ALQFM

Grade 1 2 3 4 5 6 7 8

Sm$ 0.95 0.9 0.85 0.8 0.7 0.6 0.5 Sm < 0.5Pm˛ 95–105 90–110 80–120 75–125 70–130 60–140 50–150 0–Na# 0.05 0.1 0.15 0.2 0.3 0.4 0.5 a > 0.05Quality Best Better Good Fine Moderate Common Inferiors Defective

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b ¼nPni¼1

xiyi �Pni¼1

xi

Pni¼1

yi

nPni¼1

yi2 �Pni¼1

yi2� mR

mS

� 100% (2)

PL ¼ 1

2

0BBBB@

Pni¼1

ðxi � xiÞðyi � yiÞffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPni¼1

ðxi � xiÞ2s ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPn

i¼1

ðyi � yiÞ2s � b

þPni¼1

xi

Pni¼1

xiyi

Pni¼1

yi

ffiffiffiffiffiffiffiffiffiffiffiffiPni¼1

xi2

s ffiffiffiffiffiffiffiffiffiffiffiffiPni¼1

yi2

s � 100%

1CCCCA (3)

R% ¼Pni¼1

xi

Pni¼1

yi

� mR

mS

� 100% (4)

a ¼��Rb � 1

�� (5)

3. Materials and methods3.1 Chemicals and reagents

A total of 35 batches of CLT samples (S1–S35) were supplied byShandong Xinhua Pharmaceutical Co., Ltd. (Shandong, China;Manufacturer A, providing S1–S15) and China National Phar-maceutical Co., Ltd. (Hebei, China; Manufacturer B, providingS16–S35), respectively. Individual herbs, including glycyrrhizaextract and powdered poppy capsule extractive, were obtainedfrom China National Pharmaceutical Co., Ltd. (Hebei, China).Nine reference standards, liquiritin (LQT), SMB, codeinephosphate (CON), andmorphine (MPE), were acquired from theNational Institutes for Food and Drug Control. Liquiritin apio-side (LQA), liquiritigenin (LQG), isoliquiritigenin (ISG), iso-liquiritoside (ISS), and glycyrrhizic acid (GLA) were provided byShanghai Winherb Medical Technology Co., Ltd. (Shanghai,China). In the UHPLC-ESI-Q-TOF-MS/MS analysis, methanol(HPLC grade) and acetonitrile (HPLC grade) were provided fromMerck (Darmstadt, Germany). Formic acid (HPLC grade) andammonium acetate were purchased from Yuwang ChemicalIndustry Co., Ltd. (Shandong, China). In the HPLC and UVspectroscopic analysis, acetonitrile (HPLC grade), methanol(HPLC grade), and anhydrous ethyl alcohol (HPLC grade) were


purchased from Yuwang Chemical Industry Co., Ltd. (Shan-dong, China). Sodium 1-heptanesulfonate was obtained byZhongmei Chromatographic Co., Ltd (Shandong, China).Phosphoric acid (HPLC grade) was supplied from KermelChemical Reagent Co., Ltd (Tianjin, China). Deionized waterwas puried by a Milli-Q system (Bedford, MA, USA). All theother reagents were of analytical grade.

3.2 Sample and standard preparation

The reference standards of LQT, SMB, CON, MPE, LQA, LQG,ISG, ISS, and GLA were accurately weighed separately and dis-solved in methanol. To obtain CLT solutions, ve tablets ofCLTs were milled into powder and accurately weighed. Thepowder was then extracted with 50 mL methanol/water/phosphoric acid (160 : 40 : 1, v/v/v) solution in an ultrasonicbath for 20 min. All the solutions were ltered through 0.45 mmMillipore lters (Beijing Sunrise T&D Company, China) andstored at 4 �C prior to use.

3.3 Experimental conditions

3.3.1 Instruments and MS conditions. Chemical compo-nents analysis was performed on aWaters UPLC system (WatersTechnologies Co., Ltd., Milford, MA, USA) coupled witha Waters ACQUITY UPLC HSS T3 C18 column (100 mm � 2.1mm, 1.8 mm) at 30 �C. The optimal mobile phase was composedof 2 mM ammonium acetate-aqueous solution containing 0.1%(v/v) formic acid (A) and acetonitrile (B). The solvent gradientwas set as follows: 5–25% B at 0–2 min, 25–40% B at 2–10 min,40–60% B at 10–15 min, 60–90% B at 15–18 min, 90% B at 18–30 min. The ow rate was 0.3 mL min�1, and 1 mL of the samplewas injected in to the column.

The MS analysis was achieved on a Waters Xevo G2-S Q-TOFmass spectrometer (Waters Corporation, Manchester, UK)equipped with a Spray™ ESI source in both the positive andnegative ion mode. The following operating parameters wereset: ion spray voltage, +3.0 kV for the positive ion mode and�2.0 kV for the negative ion mode; cone voltage, 80 V for thepositive ion mode and 40 V for the negative ion mode; sourcetemperature, 80 �C for the positive ion mode and 120 �C for thenegative ion mode; desolvation temperature, 200 �C for thepositive ion mode and 150 �C for the negative ion mode; conegas ow rate, 10 L h�1 for the positive ion mode and 50 L h�1 forthe negative ion mode; and desolvation gas ow rate, 600 L h�1

for the positive ion mode and 700 L h�1 for the negative ionmode; collision energy range of 30–60 for the positive ion modeand 20–40 for the negative ion mode; scanned range ofm/z 100–1200 for MS1 and 50–1200 for MS2.

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3.3.2 Instruments and HPLC chromatographic conditions.HPLC chromatographic analysis was performed on an Agilent1100 HPLC series (Agilent, USA), equipped with an onlinedegasser, an auto sampler, a low pressure mix quaternarypump, and a UV-vis DAD. Chromatographic separation wascarried out on a CAPCELL PAK C18 MG column (250 � 4.6 mm,5.0 mm) (Shiseido, Japan) at 35 �C. The mobile phase wascomposed of a 5 mM sodium 1-heptanesulfonate-aqueoussolution containing 0.1% (v/v) phosphoric acid (A) and anacetonitrile-anhydrous ethyl alcohol-aqueous solution contain-ing 3% (v/v) phosphoric acid (B; 82 : 10 : 8, v/v/v). The separa-tion was affected utilizing a linear gradient as follows: 6–18% Bat 0–10 min, 18–33% B at 10–20 min, 33–46% B at 20–32 min,46–60% B at 32–45 min, 60–78% B at 45–60 min, 78–80% B at60–65 min. The injection volume and the ow rate were 10 mLand 1.0 mL min�1, respectively. The detection wavelength wasset at 220 nm, 250 nm, 280 nm, and 344 nm.

3.3.3 UV spectroscopic conditions. UV spectra wasmeasured on an Agilent 1100 HPLC series (Agilent, USA)equipped with a UV-vis DAD over the wavelength range 190–400 nm by replacing the chromatographic column with a hollowpolytetrauoroethylene (PTFE) pipe (5000 mm � 0.18 mm i.d.from Agilent). The mobile phase A–B (50 : 50, v/v) was adoptedas the carrier, with FIA being used as the analytical principle, asshown in Fig. 1A. The parameters for the separation were set asfollows: temperature of the PTFE tube 35 �C, ow rate 0.5mL min�1, CLT sample injection volume 1 mL, wavelengthinterval 1 nm, and slit width 1 nm, respectively.

3.3.4 Antioxidant activity conditions. The DPPH radicalscavenging activity assays were performed according to Mrazeket al.15 with slight modication, as shown in Fig. 1A. Comparedwith the UV spectrum analysis, 0.127 mM DPPH solutioncarried by another Agilent 1100 HPLC pump (pump 2) was usedwith a second entrance of the PTFE pipe at 0.3 mL min�1

owrate. The mobile phase A–B (50 : 50, v/v) was delivered at a ow

Fig. 1 FIA analytical principle plot for the UV spectra (A), and 3D chromaspectra (C) and four-wavelength fusion HPLC fingerprints (D) for 35 CLT

27664 | RSC Adv., 2018, 8, 27661–27673

rate of 0.4 mL min�1 and the other conditions were set asmentioned in Section 3.3.2. Finally, the absorbance of themixture was measured by the decrease at 517 nm with a UV-visDAD aer reacting in the PTFE pipe. Then, the corrected sampleelimination ratio (SER, eqn (8)) was calculated utilizing theelimination ratio (ER, eqn (6)) corrected by a correction factor(fi, eqn (7)) to evaluate the antioxidant activity of the sample.

ER ¼ A250 � A0250

A250

� 100% (6)

fi ¼

1

35

X35i¼1

A517 � m

mi

!

A517 � m

mi

¼ 1

35�

mi

P35i¼1

A517

mi

A517

(7)

SER ¼ ER� fi ¼ 1

35� A250 � A0

250

A250

�mi

P35i¼1

A517

mi

A517

� 100% (8)

where A250, A0250 were the chromatographic peak areas of theunseparated sample before and aer reaction with DPPH at250 nm, respectively. The negative peak area of DPPH aerreaction with the sample at 517 nm was dened as A517. Also, fiwas the ratio between the average of the 35 negative peak areasand each negative peak area, where the negative peak area wascorrected by a mass correction factor. The higher the SER, thestronger the antioxidant activity of the sample.

3.4 Data analysis

MassLynx 4.1 soware (Waters, USA) was used for the UHPLC-ESI-Q-TOF-MS/MS data processing. Chromatographic ngerprintswere assessed by laboratory-developed soware (Digitized Evalua-tion System for Super-Information Characteristics of TCM Chro-matographic Fingerprints 4.0; Soware certicated no. 0407573,China). SIMCA 13.0 was applied for the data analysis.

togram plot (min � nm � absorbance) (B) for CLT samples, typical UVsamples.





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4 Results and discussion4.1 UHPLC-ESI-Q-TOF-MS/MS analysis of the CLT sample

Before the MS data analysis, an in-house formula databaseinvolving the compound name, chemical structure, molecularformula, accurate mass, and related product ions of thecompounds in the individual herbs of CLT was established bysearching a number specialist of databases, such as Reaxys(https://www.reaxys.com/), Scinder (https://scinder.cas.org),PubMed (https://www.ncbi.nlm.nih.gov/pubmed) and Elsevier(https://www.sciencedirect.com/science/bookbshsrw). Both thepositive and negative ion modes were employed in the chemicalcomposition analysis of CLT (as shown in Fig. 2). However, toavoid repetition, we mainly list the negative ions in the ESITable 1† unless some compounds could only be detected in thepositive ion mode. In total, 41 compounds were identied (asdepicted in Fig. 3), most of them originating from LRC (19avonoids and 19 saponins); only 2 alkaloids were from PPCE,the remaining ingredient was identied as SMB. Among thecompounds identied, 9 compounds (peaks 1, 2, 6, 7, 8, 11, 15,19, and 23) were unambiguously identied by comparing withthe reference standards. The other compounds were tentativelycharacterized based on the mass accuracy of their precursorions (within a 5 ppm error), tandem MS spectra, and fragmen-tation pathways, referring to the previous literature.27–44 Aera thorough literature search, 2 compounds, comprising 1avonoid (isoliquiritigenin-40-apiosyl(1/2)glucoside) and 1saponin (apioglycyrrhizin or araboglycyrrhizin), were rstidentied in CLT in the present study.

4.1.1 Identication of avonoids in the CLT sample. A totalof 19 avonoids originating from LRC were detected in the CLTsamples, comprising 11 chalcones, 4 avanones, 3 avones, and1 isoavone. Peaks 6, 7, 11, 15, and 23 were unambiguouslyattributed to LQA, LQT, ISS, LQG, and ISG by comparison withthe reference compounds.

Chalcones and avanones. In light of the structural charac-teristics and typical fragment ions in the UHPLC-MS/MS data,chalcones and avanones could be divided into two groups (asshown in Fig. 4): chalcones with R10 ¼ OH could easily trans-form to the corresponding avanone isomers. Thus, chalconesand avanones showed similar fragmentation pathways togenerate the characteristic ion at m/z 255 by elimination of anapiosyl group (Api, 132 Da), glucosyl group (Glu, 162 Da), orboth of them, and yield fragment ions at m/z 135 and 119 viaretro Diels–Alder (RDA) reaction in the negative ion mode.However, the UV spectrum could aid distinguishing them.Nevertheless, this only worked with a high purity and abundantintensity of chromatographic peaks. Liquiritigenin-40-apio-syl(1/2)glucoside, a typical avanone, was used to characterizethe fragmentation pathway (Fig. 2C). Based on these fragmen-tation patterns, the reference compounds, and related litera-ture, compounds 5, 9, 10, 11, 12, 23, 6, 7, and 15 were identiedas isoliquiritigenin-40-apiosyl(1/2)glucoside, isoliquiritigenin-40-apiosyl(1/2)glucoside, isoliquiritigenin-40-apiosyl(1/2)glucoside, isoliquiritoside, neoisoliquiritin, isoliquiritigenin,liquiritigenin-40-apiosyl(1/2)glucoside, liquiritin, and


liquiritigenin, respectively. In particular, since both A and Brings in glabrol (compounds 37) contained isopentenyl struc-tures, two fragment ions at m/z 203.0701 and 187.1118 wereproduced via RDA reaction; in the case of no hydroxyl at R10, thefragmentation patterns were dramatically different. The cleav-ages mainly occurred around the carbonyl, and the ion abun-dance depended on the stabilities of the fragments. Generally,the number of phenolic hydroxyls determined the stability ofthe structure in the negative ion mode. Licochalcone B(compound 14), possessing three hydroxyl groups, gavea deprotonated molecule [M � H]� at m/z 285.0755 andproduced predominant fragment ions at m/z 177.0181 [M � H–

C6H5O–CH3]�, 150.0313 [M � H–C7H6O2–CH3]

�, and 121.0283[M � H–C9H10O3]

� in the MS/MS spectra. Based on thesecleavage patterns, peak 30 was identied as licochalcone D.Licochalcone C (compound 33), possessing two hydroxylgroups, gave the deprotonated molecule [M � H]� at m/z337.1433 and produced fragment ions at m/z 305.1175 [M � H–

CH3OH]�, 229.0864 [M � H–C6H5O–CH3]�, 201.0907 [M � H–

C7H5O2–CH3]�, and 120.0209 [M�H–C14H17O2]

� in the MS/MSspectra. Moreover, B rings, containing the isopentenyl struc-ture, tended to strip off the neutral fragment of C4H7$ (55 Da)and produced predominantly fragment ions at m/z 146.9652(Fig. 2D). According to these fragmentation patterns, peaks 34and 35 were identied as licochalcone E and licochalcone A,respectively.

Flavones. Compound 13 (40,7-dihydroxyavone) representsthe basic skeleton of avones in licorice. The C-ring undergoesRDA fragmentation to generate fragments at m/z 135.0079(1,3A�) and 117.0334 (1,3B�). Compounds 3 and 4 were charac-terized as avone C-glucosides, which unlike avone O-glycosides have that lost the whole glycosyl segment, whichindicates a special cleavage pathway with successive or simul-taneous losses of the glycosyl group. The avone C-glucosidesgenerated the product ions by losing 30, 60, 90, and 120 Dafrom the precursor ions. Similarly, the avone C-arabinosidesproduced typical losses of 30, 60, and 90 Da, and avone C-rhamnosides showed the characteristic elimination of 44, 74,and 104 Da. For example, isoviolanthin (compound 4, ESIFig. S1A†) generated the product ions atm/z 457.1124, 383.0754,and 353.0657 from the precursor ions atm/z 577.1559, and thencleavage of the ion at m/z 253.0495 gave rise to fragments at m/z135.0072 via RDA reaction. Based on these cleavage patterns,compound 3 was identied as an isoschaoside.

Isoavones. Formononetin (ESI Fig. S1B†) is a representativeisoavone in licorice. It shared a base peak at m/z 267.0653 inthe ESI negative ion mode and was commonly observed withlosses of $CH3 (15 Da) and CO2 (44 Da). In the MS2 spectra, itproduced fragment ions at m/z 252.0420 [M � H–CH3]

�, m/z223.0393 [M � H–CO2]

�, and m/z 195.0444 [M � H–CO2–CO]�.

The RDA reaction still occurred and generated fragments at m/z135.0080 (1,3A�) and 132.0209 (1,3B�).

4.1.2 Identication of triterpene saponins in the CLTsample. Triterpene saponins are the major active ingredients inLRC, and all of them belong to oleanane-type triterpene sapo-nins. These compounds produced messy fragment ions in thepositive ion mode, except for two of them (compounds 20 and

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Fig. 2 Total ion chromatogram of CLT in the positive (A) and negative ion mode (B), the ESI-MS/MS spectrum and proposed fragmentationpathways of liquiritin apioside (C), licochalcone C (D), glycyrrhizic acid (E) in the negative ion mode and morphine (F) in the positive ion mode.

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25) that yielded conventional secondary fragment ions in theirMS2 spectra, while the others generated secondary fragmentions from the cleavages of triterpene-skeleton. The prominentlosses ofm/z 176 [GlcA–H2O], 132 [Api–H2O], 44 [CO2], 18 [H2O],

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193 [GlcA–H]�, and 351 [2GlcA–H2O–H]� were highly charac-teristic for identication of the triterpene saponins. Successivelosses of sugar moieties were helpful for prediction of the sugarnumbers and sequences.





Fig. 3 Chemical structures of 41 identified compounds in CLT (unless otherwise noted, the substituent groups (R) are H).

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By comparison with the authentic compound, compound 19was unambiguously attributed to glycyrrhizic acid, which wasused to illuminate the fragmentation pathway as a typical tri-terpene saponin in LRC (Fig. 2E). The [M � H]� at m/z 821.3976was fragmented into characteristic ions at m/z 645.3629, m/z469.3314, and m/z 351.0567, corresponding to the [M � H–


GlcA]�, [M�H–2GlcA]�, and [2GlcA–2H2O–H]�, respectively. Byexploring the literature and these fragmentation patterns,compounds 16–18, 20–22, 25–29, 31, 32, 36, and 38–41 wereidentied as licorice saponin G2, yunganoside K2 or its isomer,yunganoside K3 or its isomer, an isomer of licorice saponin B2,licorice saponin K2, licorice saponin H2, licorice saponin C2,

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Fig. 4 Strategy for rapid structural identification by MS/MS spectra in CLT.

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apioglycyrrhizin or araboglycyrrhizin, licorice saponin B2,apioglycyrrhizin or araboglycyrrhizin, an isomer of licoricesaponin B2, glycyrrhetinic acid glucuronide, isomer of glycyr-rhetinic acid glucuronide, glycyrrhetinic acid hydroxylate, 18-a-glycyrrhetic acid, 18-b-glycyrrhetic acid, 3-oxoglycyrrhetic acid,and 11-deoxoglycyrrhetic acid, respectively.

4.1.3 Identication of alkaloids in the CLT sample. 2alkaloids (codeine and morphine) primarily from PPCE weresuccessfully identied with the reference standards utilizing thepositive ion mode. Their spatial molecular structures were notstable in the collision, resulting in a variety of bond breakingways and forming abundant fragment ions. There was only onedifferent functional group (hydroxyl, methoxy) in the chemicalstructure between morphine and codeine. By MS2 analysis ofthe molecular ions at m/z 286. 1440 [M + H]+ and m/z 300.1601[M + H]+, it was shown that they had similar cracking rules.Therefore, here, morphine is used to illuminate the fragmen-tation pathway as a typical alkaloid in PPCE (Fig. 2F).

The loss of H2O produced m/z 268.1336, followed by a loss ofCH4 or C2H3N to produce fragments m/z 252.1024 or 211.0749,respectively. The further fragmentation of ionm/z 211.0749 gaverise to other fragments. A key ion wasm/z 229.0853 aer the lossof the nitrogen bridge from the parent ionm/z 268.1336 and thiswas subsequently fragmented to ions m/z 227.0706, m/z201.0907, and m/z 211.0749 aer the losses of H2, CO, and H2O,respectively. The loss of H2O from m/z 201.0907 led to ion m/z183.800, the other key ion, which was subsequently fragmentedto ions m/z 181.0646, m/z 165.0700 (base peak with a highlyconjugated frame), and m/z 153.0695 aer the simultaneous orsuccessive losses of H2, CO, and H2O.

4.1.4 Identication of sodium benzoate in the CLT sample.Based on the analysis of the authentic compound, compound 8was unambiguously attributed to sodium benzoate. Sincesodium benzoate was an acid in the negative ionizationmode, itprovided superior sensitivity in comparison to the positive

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mode. An [M � H]� at m/z 121.0287 was found in the negativeion mode, which could successively lose CO2 (44 Da) to forma fragment at m/z 77.0373 (C6H5).

According to above studies, it was concluded that thechemical components in the CLT samples mainly came fromthe principal individual herb, i.e., LRC (consisting of avonoidsand triterpene saponins), PPCE (consisted of alkaloids), andSMB. This information about the structures of the chemicalcomponents in CLT could help in developing research strate-gies for bioactivity and QC studies.

4.2 UV spectroscopic/HPLC ngerprint analyses

4.2.1 Method validation of the ngerprint analysis. SampleS1 as described in Section 3.2 was used to perform the followingexperiments. Instrument precision was determined by sixreplicate injections of a single sample solution. Methodrepeatability was validated by analyzing six individual samplesolutions utilizing the same experimental procedure. Samplestability was assessed by analyzing the same sample solutionstored at room temperature for 0, 2, 4, 8, and 12 h.

In the UV ngerprint analysis, unseparated chromatograms(single chromatographic peak collected within 1 min) at 250 nmand UV spectra of samples in the region 190–400 nm wererecorded. A typical 3D chromatogram plot of the CLT sample(S1) is shown in Fig. 1B. The retention time (RT) and the peakarea (RA) with an unseparated chromatogram were used toestimate the repeatability, precision, and stability. The obtainedresults showed that the RSDs of RT and RA were all less than2.0% for the precision, repeatability, and stability validation.

In the HPLC ngerprint analysis, the average linear quali-tative similarity (SL) and average linear quantitative similarity(PL) of the HPLC ngerprints was used to estimate the precision,repeatability, and stability. The obtained results showed that,for precision, the RSDs of SL and PL were less than 0.2% and0.8%; for repeatability, the obtained value did not exceed 0.3%





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and 1.1%; for stability, the RSDs were less than 0.7% and 1.6%.Thus, these results demonstrated that the developed UV spec-troscopic and HPLC methods met the ngerprint analysisrequirements for the CLT samples.

4.2.2 Sample quality evaluation based on the UV nger-prints. UV RFP was constructed by taking the average of the 35corresponding spectra. As shown in Fig. 1C, the UV ngerprintsof the 35 CLT samples were very similar. Thus, the subtlespectral differences needed to be characterized by ALQFM. TheSL, PL, and a values of the UV ngerprints of the 35 CLT sampleswere computed by importing all the UV spectral data into the in-house soware mentioned above, and the obtained results areshown in Table 2.

For the UV ngerprints, the qualitative parameters SL anda values of the 35 CLT samples were, respectively, above 0.979and below 0.021, illustrating that all the CLT samples hadsimilar chemical compositions. Based on the qualitativeparameters SL and a, the quality grades of the 35 CLT samples

Table 2 The evaluation results assessed by ALQFM and SER for experim

Sample

UV Fusion

SL PL% a Grade SL PL

S1a 0.997 112.9 0.008 3 0.959 118S2a 0.992 104.8 0.000 1 0.961 112S3c 0.997 104.1 0.009 1 0.934 110S4a 0.997 111.0 0.008 3 0.956 120S5b 0.997 106.7 0.004 2 0.962 114S6a 0.995 106.0 0.002 2 0.953 114S7b 0.996 105.3 0.000 2 0.951 116S8a 0.994 111.0 0.004 3 0.960 116S9b 0.996 106.1 0.001 2 0.950 120S10a 0.986 107.6 0.006 2 0.949 119S11a 0.996 106.5 0.005 2 0.956 119S12c 0.985 110.8 0.001 3 0.884 127S13c 0.991 111.8 0.015 3 0.856 124S14a 0.990 108.0 0.021 2 0.899 118S15c 0.991 105.6 0.012 2 0.843 128S16a 0.996 90.3 0.005 2 0.886 66S17a 0.997 95.8 0.007 1 0.881 67S18a 0.996 93.0 0.006 2 0.891 66S19a 0.996 87.2 0.001 3 0.907 67S20b 0.996 94.0 0.007 2 0.906 67S21a 0.996 91.4 0.007 2 0.924 74S22a 0.996 87.8 0.009 3 0.902 75S23a 0.979 84.6 0.014 4 0.922 70S24a 0.989 90.2 0.011 2 0.924 75S25b 0.996 92.4 0.007 2 0.892 76S26a 0.996 89.6 0.020 3 0.921 82S27a 0.996 94.5 0.015 2 0.918 80S28a 0.995 94.1 0.014 2 0.930 79S29a 0.994 94.1 0.016 2 0.943 83S30b 0.991 88.7 0.017 3 0.934 83S31a 0.987 101.3 0.001 1 0.970 95S32a 0.999 104.8 0.005 1 0.973 95S33a 0.998 101.2 0.002 1 0.971 93S34a 0.998 99.7 0.010 1 0.969 95S35b 0.976 102.8 0.001 1 0.965 100

a Used for the calibration model. b Used for the prediction model. c Outli


should have the highest quality. In fact, only 8 samples (S2, S3,S17, and S31�S35) met the level of grade 1, while the remainingones were in the range of grade 2–4 in combination with PL froma quantitative perspective. For example, 17 samples (S5�S7,S9�S11, S14�S16, S18, S20, S21, S24, S25, and S27�S29) werejudged as grade 2 with PL in the range 94.1–106.7%; 9 samples(S1, S4, S8, S12, S13, S19, S22, S26, and S30) were judged asgrade 3 with PL in the range 88.7–112.9%; 1 sample (S23) wasjudged as grade 4 with PL 84.6%, respectively. The above resultsdemonstrated that, although the qualitative evaluation (SL anda) was important, the further quantitative assessment (PL)should not be ignored. As a quantitative parameter, PL describesthe overall ingredient content in the samples. Therefore, PL hasa great potential to be associated with the medicinal efficacy inclinical situations. In general, samples above grade 5 were rec-ommended as the qualied ones. Accordingly, in this study, thequalities of the 35 CLT samples were all judged as qualied withthe UV ngerprints.

ental and predicted values

Pred (SCR) Var (SCR) REd(%)% a Grade

.7 0.037 4 42.9020 43.3005 �0.92

.8 0.053 3 44.5328 44.8081 �0.61

.5 0.037 3

.1 0.049 5 41.3406 43.0298 �3.93

.9 0.048 3 41.6980 41.3589 0.82

.8 0.038 3 40.8171 41.9347 �2.67

.7 0.049 4 41.1791 40.6103 1.40

.5 0.048 4 41.7202 41.5415 0.43

.8 0.052 5 41.3713 41.4627 �0.22

.1 0.031 4 41.4828 39.7119 4.46

.0 0.040 4 41.3830 39.8654 3.81

.1 0.011 5

.9 0.021 5

.4 0.080 4 41.4751 41.5693 �0.23

.8 0.021 5

.8 0.061 6 33.9911 34.7101 �2.07

.8 0.056 6 33.8583 33.0766 2.36

.8 0.088 6 34.4281 33.0232 4.25

.1 0.071 6 34.4853 34.8612 �1.08

.4 0.074 6 34.0604 33.4956 1.69

.6 0.029 5 36.3014 36.8928 �1.60

.8 0.012 5 36.5263 36.3241 0.56

.8 0.040 5 36.6813 37.6926 �2.68

.2 0.024 5 36.8588 36.1093 2.08

.1 0.031 5 36.6918 37.1300 �1.18

.9 0.004 4 38.4024 38.8688 �1.20

.9 0.010 4 38.6008 37.8988 1.85

.9 0.017 5 37.3817 37.1949 0.50

.2 0.011 4 37.5003 36.5895 2.49

.2 0.013 4 38.1066 38.3294 �0.58

.7 0.060 2 35.4962 34.6755 2.37

.2 0.052 2 35.5709 37.1779 �4.32

.0 0.034 2 35.1585 35.8153 �1.83

.8 0.052 2 35.3569 35.6429 �0.80

.7 0.059 2 35.5584 36.2390 �1.88

ers. d RE, relative error.

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4.2.3 Integrated evaluation based on the fusion nger-prints. According to the chemical structural analysis (Fig. 3) andthe maximum UV absorption (ESI Table 1†), the strong UVabsorption bands (Fig. 1B and C) of the CLT samples appearedat 220 nm, 250 nm, 280 nm, and 344 nm, corresponding to theavonoids, triterpene saponins, alkaloids, and sodiumbenzoate, regarded as the major substances in CLT (asdescribed in Section 4.2.2). Therefore, fusion ngerprints of thefour wavelengths (220 nm, 250 nm, 280 nm, and 344 nm) couldrealize the overall assessment of the CLT samples by importingthe signal data into the in-house soware mentioned above.The integrated quality grades (Table 2) could be classied byALQFM (Table 1).

For the fusion ngerprints, the acceptable SL and a values ofthe 35 CLT samples were above 0.843 and below 0.088, respec-tively, indicating that they were similar in the number anddistribution of chemical components, and there was littlevariability between the samples. The acceptable PL values wereset in a wider range (70.0–120.0%) and could exactly discrimi-nate the CLT samples from every piece of content of thengerprints, but actually SL and a were disabled for this func-tion. For example, S2, S3, S5, and S6 should be the grade 2 basedon the parameters SL and a. However, they were the highergrades in combination with PL. S16–S20 had unqualied inte-grated grades (grade 6) due to the much lower contents ofcomponents, while the remaining 30 samples had qualiedones (grades 1–5, where there was no judgment sample in grade1; grade 2 with PL in the range 93.0–100.7%: S1–S35; grade 3with PL in the range 110.5–114.9%: S2, S3, S5, and S6; grade 4

Fig. 5 PCA scores scatter plot for the UV spectra (A) and fusion fingerprinOPLS standardized regression coefficient plot (D).

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with PL in the range 80.9–119.1%: S1, S7, S8, S10, S11, S14, S26,S27, S29, and S30; grade 5 with PL in the range 70.8–128.8%: S4,S9, S12, S13, S15, S21–S25, and S28). ALQFM could balance theeffects of the large and small peaks; therefore, the qualities ofthe 35 CLT samples could be more accurately distinguishedwith the HPLC ngerprints.

4.2.4 Comparison of the two evaluation methods based onthe UV/HPLC ngerprints. In order to carry out a detailedinvestigation of the distinguishing ability of the HPLC and UVspectroscopic results, principal component analysis (PCA),a well-known chemometrics method, was performed utilizingthe SIMCA 13.0 soware. The four main UV absorption bands(220 nm, 250 nm, 280 nm, and 344 nm) in Fig. 1B and C and thepeak area of the four wavelengths fusion HPLC ngerprintpeaks (Fig. 1D) were used as the input data. Both HPLC and UVspectroscopic PCA models were constructed utilizing a four-component model with a total variance of 84.5% and 92.9%explained (HPLC, PC1 ¼ 56.3%, PC2 ¼ 15.1%, PC3 ¼ 7.5%, andPC4 ¼ 5.6%; UV, PC1 ¼ 75.8%, PC2 ¼ 13.0%, PC3 ¼ 2.6%, andPC4 ¼ 1.5%), respectively, indicating that the UV spectra re-ected less information than HPLC; however, it is undeniablethat the less information, the easier it can be described by thePCA model.

The PCA score plot in Fig. 5A and B revealed that mostsamples fall into one cluster except for S12, S13, and S15 inHPLC. The PCA results were in good agreement with the ALQFManalysis, providing strong evidence that the quality of S12, S13,and S15 might be different from the other samples. In fact, theinteresting PL value of S12, S13, and S15 were the highest among

t (B), measured versus predicted values for the calibrationmodel (C) and





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the CLT samples, which just better states why these wereconsidered outliers in the PCA score. Then, the 35 CLT samplesin the PCA score plot for the UV and HPLC methods, excludingthe outliers, could be clearly divided into two clusters marked asgroup 1 and 2, respectively. Group 1 (S1–S15, from manufac-turer A) was obviously different from group 2 (S16–S35, frommanufacturer B). It should be noted that the samples in eachcluster were more concentrated in Fig. 5B than Fig. 5A, indi-cating that UV spectral ngerprinting could be rapidly detectedand evaluated, and that HPLC ngerprint analysis was recom-mended for a more accurate assessment. Consequently, fromthe PCA results, the products from the same manufacturer hada relatively good quality consistency, while the products amongdifferent manufacturers exhibited large differentiation.

Compared with the quality grades assessed by the UVngerprinting method, the fusion ngerprints results exhibitedsome uctuations and even greater differences, and this couldbe attributed to two different analytical principles, whichmainly reected the features of the separation and unseparatedchemical ingredients in the CLT samples, respectively. Forexample, S16, S17, S18, S19, and S20 had an unqualied sepa-rated quality (grade 6 in the HPLC method); however, they hadbetter unseparated ones (grades 2, 1, 2, 3, 2 in the UV method,respectively). The fusion HPLC ngerprints combined with a UVngerprints assessment strategy thus provided a feasible andreliable means to monitor the quality consistency of the CLTsamples, which was very necessary to avoid a bias caused by theUV ngerprinting method.

4.3 Relationship between the fusion ngerprints andantioxidant activities in vitro

According to the existing literature, avonoids, triterpenesaponins, and alkaloids possess excellent antioxidant abili-ties.45–47 Simultaneously, combined with the chemical compo-nents in ESI Table 1,† the antioxidant abilities of the CLTsamples might be due to the presence of chemical constituents,such as avonoids, triterpene saponins, and alkaloids. Thisinformation encouraged us to investigate the ngerprint–effi-cacy relationship between the fusion ngerprints and the anti-oxidant activities. The fusion ngerprints and antioxidantactivities in vitro were investigated under the conditionsdescribed in Sections 3.3.2 and 3.3.4, respectively. Typicalfusion chromatograms with 68 co-possessing ngerprints forthe 35 CLT samples are shown in Fig. 1D. Correlation analysisbetween the SER values (as the Y variables) and the HPLCngerprints (as the X variables) was performed by the OPLSmethod.

Aer omitting the four outliers (S3, S12, S13, and S15) basedon the t[1]–t[2] score plot, the remaining CLT samples wererandomly divided into two groups (Table 2) of the calibration set(24 samples) to establish a validation set (7 samples) to validatethe OPLS model. The established calibration model (Fig. 5C)achieved an explained variance (R2) of 91.84%, a predictiveability (Q2) of 87.00%, and a root mean square error of esti-mation of 0.9851, respectively, indicating that the presentmodel was excellent. The validation set was used to assess the


activity prediction of the obtained model. A satisfactory resultwith an explained variance (R2) of 97.49% and a root meansquare error of prediction (RMSEP) value of 0.4584 was ob-tained, indicating the calibration model possessed a wellpredictive ability. The predicted vs. measured SER values forboth the validation and calibration models are shown in Table2, where no signicant difference could be observed for the CLTsamples. As shown by the standardized regression coefficientsplot (Fig. 5D) of the calibration model, 38 peaks (2–5, 8, 10, 12,14, 17, 19, 20, 23–26, 28, 30, 31, 34, 36, 41, 43, 44, 46, 48–54, 56,57, 61–64, and 70) out of 70 ngerprints in the fusion chro-matogram were positively correlated, while the remaining peakswere negatively correlated with SER, indicating that themajorityof the chemical components in the CLT samples possessedantioxidant abilities. Furthermore, the peaks 20, 25, 26, 28, 31,36, 41, 51, and 54 in the fusion ngerprints were identied asMPE, LQA, LQT, CON, SMB, ISS, LQG, ISG, and GLA, respec-tively, by comparing the retention time and the online UVspectra with reference standards.

5 Conclusions

In this paper, a reliable and sensitive UHPLC-ESI-Q-TOF-MS/MSmethod was performed for identication of the chemicalproling of CLT samples. A total of 41 compounds, including 19avonoids, 19 saponins, and 2 alkaloids, were identied ortentatively deduced by comparing their retention times and theMS spectrometry data in the literature. Based on UHPLC-ESI-Q-TOF-MS/MS analysis and the UV absorption, the four main UVabsorption bands (220 nm, 250 nm, 280 nm, and 344 nm) of theCLT samples could basically reect the overall information ofthe CLT samples. Accordingly, the rapid UV spectroscopicngerprints and the accurate multi-wavelength (220 nm,250 nm, 280 nm, and 344 nm) fusion HPLC ngerprints wereintegrated in equal weight to reect the overall characteriza-tions of the CLT samples. In the ngerprint assessments,ALQFM with qualitative and quantitative assessment advan-tages was recommended and established for scientic CLTquality differentiation rst, and it could overcome the defects inthe quantitative criteria lacking in evaluation methods.According to the integrated UV spectroscopic ngerprints andmulti-wavelength fusion HPLC ngerprints as well as ALQFM,the quality consistency of the 35 CLT samples from twomanufacturers exhibited almost the same results, indicatingthat the combination of the two ngerprinting methodsprovided a reliable means to monitor the quality consistency ofthe CLT samples. Moreover, PCA was applied to explore thediscriminating ability of the two ngerprinting methods, andthe results demonstrated that samples from the same manu-facturer had a relatively good quality consistency and that theUV spectra provided less information than from HPLC. Tofurther investigate the antioxidant potential of CLT, the freeradical scavenging capacity was rapidly assessed by FIA, whichwas suitable for batch samples in antioxidants screening. Inaddition, the ngerprint–efficacy relationship between multi-wavelength fusion HPLC ngerprints and antioxidant activi-ties was conducted utilizing OPLS, providing important

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supplemental bioactivity information for CLT QC. This studyreported important clues for the further pharmacological studyof CLT and offered a rapid, holistic, and scientic analyticalstrategy for CLT/TCM QC, which could play an important role inCLT practical production.

Conflicts of interest

There are no conicts to declare.

Acknowledgements

This work was supported by National Natural Science Founda-tions of China (81573586).

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